Lung elastin degradation occurs with the development of pulmonary emphysema in patients with COPD related to smoking or alpha-1 antitrypsin deficiency. COPD a lengthy, chronic, progressive disease, is debilitating and often non-reversible. COPD is a growing and costly problem. COPD currently affects over 18 million Americans and is the fourth leading cause of death in the US. According to the World Health Organization in 2007, 210 million people worldwide have suffered from COPD and 3 million of those died in 2005 alone68. According to the current trends, WHO estimates that it will become the third leading cause of death worldwide by the year 2030.
Although a number of different mechanisms may be responsible for the loss of alveoli in pulmonary emphysema, damage to elastic fibers is a significant factor in the pathogenesis of this disease1,2. These fibers are responsible for the mechanical recoil that facilitates the expiration of air from the lungs, and their breakdown can lead to alveolar distention and rupture.
Elastin fibers are part of the extracellular matrix and are an essential structural component of lung, skin, and blood vessels. Desmosine (D) and Isodesmosine (I) are two unique pyridinium amino acids that serve as crosslinking molecules binding the polymeric chains of amino acids into the 3D network of elastin33-35. The degradation of elastin-containing tissues that occurs in several widely prevalent diseases, such as atherosclerosis, aortic aneurysms, cystic fibrosis and COPD which includes pulmonary emphysema etc., have been associated with increased excretion in the urine of peptides containing these two pyridinium compounds36-44. As noted above, in lung, elastin degradation occurs with the development of COPD related to smoking or related to α1-antitrypsin deficiency (AATD)45,46.
Due to the importance of elastic fiber injury in pulmonary emphysema, a number of therapeutic approaches have focused on protecting this extracellular matrix component from degradation by elastases and other injurious agents3,4. However, determining the effectiveness of such treatment is often difficult because of the lack of sensitive, real-time indicators of successful therapeutic intervention. Both pulmonary function tests and high-resolution computerized tomography (HRCT) require prolonged time intervals to assess the potential benefits of a therapy, while more sensitive markers such as proinflammatory cytokines lack the necessary specificity to determine efficacy5-9. One possible solution to this problem is measurement of elastic fiber breakdown products themselves.
Desmosine and isodesmosine, the crosslinking amino acids present only in elastin in the human, offer the prospect of assessing elastin degradation in disease by their measurement in certain body fluids. Thus far, D and I have been measured in urine of patients with COPD and found to be statistically significantly elevated above normal controls. One study demonstrated the daily variability of excretion of desmosine and isodesmosine and did not show a statistically significantly elevated excretion of these amino acids in patients in 24-hour collections. In this same study, statistically significantly increased excretion of desmosine and isodesmosine was found in patients with cystic fibrosis.
Various techniques including RIA47,48, HPLC49-51, and capillary zone electrophoresis52,53 have been utilized for the analysis of urinary D and I. Measurements in subjects with COPD show increased levels of D and I in acid hydrolyzed sputum and plasma, along with elevated free D and I in urine without acid hydrolysis11,54.
Recent advances in detecting the elastin-specific amino acid crosslinks desmosine and isodesmosine have greatly increased the sensitivity and specificity of this test procedure10-14. Levels of D and I in urine and plasma have been shown to correlate with physiological and radiological measures of COPD12.
Peptides of elastin have been measured in plasma by radioimmunoassay (RIA) and found to be elevated in patients with COPD. Because of variability of the specificity of antibodies to elastin peptides in such RIAs, however, quantitation of such peptides has varied among various studies. Advances have been made in the ability to measure D and I in certain complex biological samples using mass spectrometry. For example, the LC/MS analysis which provides increased sensitivity and specificity may be an important method for biomarker analysis of elastin degradation in disease55.
Three LC/MSMS analyses of D and I have been reported56-58. These studies have been limited to the analysis of D and I content in urine or mouse lung hydrolysates. As we have shown in the study of D and I in COPD11,54 and a study on the effect of Tiotropium therapy on D and I levels in COPD10, it is recommended that D and I levels be evaluated in other body fluids as well; such as sputum, plasma, or bronco alveolar lavage fluid. An accurate and reproducible quantification of D and I in several body fluids may be more useful to characterize elastin degradation in disease and to follow the course of disease and therapies59. In addition, two recent FDA workshops sponsored by the COPD Foundation held in May, 2009 and January, 2010 have called for standardization of the analysis to provide practical clinical biomarkers for COPD.
Active smoking is the most important modifiable risk factor for COPD69. In comparison to previous years, overall less people smoke now, however, there has been a disturbing increase in active smokers in the under-30 age group68. Once active smoking was established as having detrimental health effects on lung disease, the focus turned to the possible adverse effects of passive smoking—the atmospheric exposure to second hand smoke.
Second Hand Smoke exposure (SHS) has been implicated as a risk factor in many diseases including asthma, bronchitis, and coronary artery disease70. As a result, there is a worldwide campaign to eliminate passive smoking from the environment71. Second hand smoke increases the risk of heart disease in adults72 and has been shown to increase the inflammatory state73. Flouris et al. demonstrated an increase in inflammatory cytokines in individuals who had never smoked who were exposed to only one hour of second hand smoke, and these cytokines remained elevated for three hours after the exposure ceased70. In the Attica study, inflammatory cytokines were elevated from chronic exposure to second hand smoke for extended periods, and the levels were similar to these of the active smokers74.
Given the deleterious effects of passive smoking on overall health, it is reasonable to consider its detrimental effects on lung parenchyma itself. Studies of subjects exposed heavily to second hand smoke, i.e. bar workers, flight attendants, have shown an increase in lung cancer, COPD, bronchitis, and asthma exacerbations75. In the same studies, subjective health improved within one month of a clean air environment and objective improvement was seen within two months. There are studies demonstrating the effect of second hand smoke exposure on biomarkers of inflammation in patients with cardiovascular as well as pulmonary disease76-78. However thus far, no studies have evaluated the effect of second hand smoke exposure on tissue matrix proteins.
In view of the foregoing, there is a need for methods of accurately detecting and measuring elastin components, such as desmosine, isodesmosine or combinations thereof, for the purpose of diagnosing and/or treating COPD, chronic bronchitis, emphysema, refractory asthma, and other related diseases and/or monitoring patients with such diseases and/or who are exposed to, e.g., SHS. Similarly, there is a need for methods of validating whether a candidate compound is effective to treat, prevent, or ameliorate the effects of a disease characterized by elastic fiber injury.